Isolation of sperm cells from other biological materials using microfabricated devices and related methods thereof

ABSTRACT

The present invention relates to cell separation using microfabricated devices. In particular, the present invention provides methods and devices for separation of sperm from biological materials, such as other cells and molecular species, in a cell mixture in a microfabricated device through the use of electroosmotic flow, electrophoretic mobility, pressure gradient, differential adhesion, and/or combinations thereof.

This application claims priority to Provisional Patent Application No.60/427,734, filed Nov. 20, 2002.

FIELD OF THE INVENTION

The present invention relates to cell separation using microfabricateddevices. In particular, the present invention provides methods anddevices for separation of sperm from biological materials, such as othercells and molecular species, in a cell mixture in a microfabricateddevice through the use of electroosmotic flow, electrophoretic mobility,pressure gradient, differential adhesion, and/or combinations thereof.

BACKGROUND OF THE INVENTION

The use of DNA typing to verify and often convict suspects in sexualcrime cases relies on the separation of the perpetrator DNA from that ofthe victim. The perpetrator DNA is most easily obtained from sperm cellscollected on vaginal swabs, taken in the routine collection of sexualassault evidence. The majority of cells collected on such swabs areepithelial cells from the victim, however, and these cells must beseparated from the sperm cells before DNA from the sperm cells can berecovered and STRs amplified for analysis by capillary electrophoresis(or other analytical methods). Effective separation of the victim's andperpetrator's DNA, combined with the ensuing preparatory/analysis steps,are time- and labor-intensive processes. At the present time, this iscarried out by chemical means involving differential lysis of the cellscollected on the vaginal swab. The multistep procedure begins by lysingthe epithelial cells while still adsorbed to the cotton swab. Duringthis time, the intact sperm cells (mainly heads since the tails havebeen degraded) are desorbed from the cotton swab, collected and thenlysed for DNA extraction using a Microcon™ concentration step or othermethods known in the art.

The multistep nature of this current method affords it the samedisadvantages from which many conventional isolation methodologiessuffer. First, the time-consuming and labor-intensive proceduretranslates into cost-ineffectiveness. Second, extensive sample handlingpresents opportunities for loss of biological material, which may beproblematic if only small amounts of starting material are available. Inaddition, extensive sample handling increases the chance ofcontamination with exogenous DNA, which can complicate interpretation ofthe results.

One possible solution to the conventional, time-consuming differentialextraction could be provided by microminiaturization of the analyticalmethodology in an embodiment that allowed for cell sorting to beexecuted rapidly and efficiently. Much progress has been made developingmicrochip-based analytical systems to carry out simple processes. In theearly stages, a number of groups demonstrated the analytical power ofmicrochips for carrying out fast separations (Harrison et al. Towardsminiaturized electrophoresis and chemical analysis systems on silicon:an alternative to chemical sensors. Sensors and Act. B. 10:107-116,1993; Manz, A., Graber, N., Widmer, H. M. Miniaturized Total ChemicalAnalysis Systems: A Novel Concept for Chemical Sensing., Sensors andActuators, B1. 8:244-248, 1990; and Jacobson et al. IntegratedMicrodevice for DNA Restriction Fragment Analysis. Anal. Chem. 199668:720-723). Patents have also been issued for these microfluidicdevices.

U.S. Pat. No. 5,486,335 to Wilding et al. discloses devices and methodsfor detecting the presence of a preselected analyte in a fluid sample.The invention provides a device comprising a solid substrate, typicallyon the order of a few millimeters thick and approximately a 0.2 to 2.0centimeters square, microfabricated to define a sample inlet port and amicroscale flow system. A sample is passed through the microfabricateddevice, and the restriction or blockage of flow through the flow systemis detected as a positive indication of the presence of the analyte. Thedevice may be adapted for operation in conjunction with a pump, forexample, to induce flow of a sample through the flow system.

Despite the laxity in the field that fast separations are adequate, ourexperience with clinical diagnostics indicates that sample preparationwill have to be integrated with electrophoresis in order for thistechnology to be fully exploited. Therefore, the present inventionaddresses a key sample preparation step, cell sorting, in cell analyses,especially for forensic analyses.

Other groups addressing the cell-separation problem use anantibody-based separation scheme. Eisenberg et al. (unpublished report)uses magnetic beads, to which sperm-specific antibodies are attached.There may be numerous problems associated with this approach including:clogging of the column by the large numbers of epithelial cells in a“real-world” sample, inability to integrate the cell separation withother microminiaturization analyses, expense of materials, and numeroussteps still required to result in PCR-ready DNA. A reliable separationmay not result using the antibody/magnetic bead approach due toextensive clogging of the column. The present invention described hereinovercomes the shortfalls of the conventional procedures as well as thisantibody/magnetic bead capture system. The non- or low-affinity basedseparation described utilizes the differing physical and biologicalproperties of the sperm and epithelial cells to effect a separation.

Microfabricated devices have recently been developed for cellseparations and transport. Kricka et al. (Applications of amicrofabricated device for evaluating sperm function. Clin Chem.39(9):1944-7, 1993) used a microfabricated device for theelectrophoretic separation of live and dead sperm based upon theirdifferences in surface charge.

U.S. Pat. Nos. 5,296,375 and 5,427,946, both to Kricka et al., disclosesdevices and methods similar to Wilding et al. above for clinicalanalysis of a sperm sample. In one embodiment, a sperm sample is appliedto the inlet port, and the competitive migration of the sperm samplethrough the mesoscale flow channel is detected to serve as an indicatorof sperm motility. In another embodiment, the substrate of the device ismicrofabricated with a sperm inlet port, an egg nesting chamber, and anelongate mesoscale flow channel communicating between the egg nestingchamber and the inlet port. In this embodiment, a sperm sample isapplied to the inlet port, and the sperm in the sample are permitted tocompetitively migrate from the inlet port through the channel to the eggnesting chamber, where in vitro fertilization occurs. The devices may beused in a wide range of applications in the analysis of a sperm sample,including the analysis of sperm morphology or motility, to assess spermbinding properties, and for in vitro fertilization.

Li and Harrison (Transport, manipulation and reaction of biologicalcells on-chip using electrokinetic effects. Anal. Chem. 69: 1564-1568,1997) showed the transport (not separation) and lysis of E. Coli, yeast,and canine erythrocytes in a microchip exploiting electrokineticeffects. Using electric fields of 100-600 V/cm, cells were directed fromthe loading reservoir to the waste or outlet reservoirs of themicrodevice.

Fu et al. (A microfabricated fluorescence-activated cell sorter. NatureBiotech. 17:1109-1111, 1999; and An integrated microfabricated cellsorter. Anal Chem. 74 (11):2451-2457, 2002) developed a microfabricatedfluorescence-activated cell sorter. This system requires that the sortedcells be fluorescently labeled, by means of expression of greenfluorescent protein or in some other manner. This method of cell sortingrequires interrogation/identification of the particle, and subsequentvalving of the flow to direct the cell into the correct reservoir on themicrodevice.

U.S. Pat. No. 6,193,647 to Beebe et al. discloses a microfluidic embryohandling device and method in which biological rotating of embryos issimulated. Fluid flow is used to move and position embryos withoutassistance of electrical stimulus or other means which may produceundesired heating of biological medium used as the fluid fortransporting and position. The device provides an excellent simulationof biological conditions and may be used for culturing, sorting,testing, evaluating, fertilizing and other similar typical handlingoperations. No cell separation is disclosed in this patent.

Separation and identification of various bacteria have been shown byArmstrong et al. (Rapid identification of the bacterial pathogensresponsible for urinary tract infections using direct injection CE.Anal. Chem. 72:4474-6, 2000; and Separating microbes in the manner ofmolecules: 1. Capillary electrokinetic approaches. Anal. Chem. 71,5465-5469, 1999) using conventional capillary electrophoresis. Armstronget al. separated and identified E. coli and Staph. Saprophyticus, whichare bacterial pathogens commonly responsible for urinary tractinfections, by using poly(ethylene) oxide as a sieving matrix. In 2001,Armstrong et al. used conventional capillary electrophoresis for theseparation of various bacteria from yeast. The microbes were detectedusing laser-induced fluorescence and, therefore, required staining witha fluorescent dye. They used this separation/detection method toevaluate cell viability using a commercially-available viability stainand detecting the difference in fluorescence emission.

SUMMARY OF THE INVENTION

The speed and efficiency of the conventional differential extractionprocedure warrants improvement by the micro-miniaturization of cellsorting. A stand-alone microdevice for rapidly sorting sperm cells fromepithelial cells and extracting DNA would improve DNA analysis in thecrime laboratories by reducing the cost of analysis through improvedspeed, reduced reagent consumption, decreased technician time, reducedsample handling-induced contamination, and ease of automation. Thepresent invention provides a novel method and device for separation ofsperm and epithelial cells on a microdevice. A separation method thatdoes not require a high affinity interaction with the cells but,instead, one that exploits electrophoretic mobility, electroosmoticflow, pressure-based flow and/or combination thereof is exploited. Thispresent invention utilizes the differential physical and biologicalproperties of the cells, such as their propensity for adhesion, specificgravity, cell surface charge, and size. Two important aspects of such acell separation mechanism separation are, but not limited thereto, themagnitude of the flow, which can be controlled by a number ofmechanisms, such as electrophoretic mobility, electroosmotic flow,pressure gradient (pump as well as gravity), and/or combinationsthereof, as well as the surface properties of the channel walls. Thepresent invention provides techniques for the isolation of sperm cellsfrom biological materials, preferably other cells and molecular species,most preferably epithelial cells, for forensic applications usingmicrofabricated devices.

In a further embodiment, the present invention is used to isolate spermfrom either other cells or other biologically-derived molecular speciesenables sperm to be concentrated in smaller volumes. This effect couldfind utility with in vitro fertilization applications. Beebe et al. hasshown that human eggs (oocytes) can be manipulated in microfabricateddevices in processes pertinent to in vitro fertilization. The devicedescribed herein for sperm cell isolation could be utilized to enhancethe concentration (number) of sperm in the collection chamber. One couldenvision how the presence of an oocyte in the collection chamber wherean enhanced concentration of sperm are collected, might improve theefficiency of in vitro fertilization.

In a further embodiment, the present invention is used to isolate spermfrom either other cells or other biologically-derived molecular speciesenables sperm quantitation. This could be accomplished with a number ofon-line counting sperm approaches as the migrate through themicrochannel to the collection reservoir. Included in these means wouldbe optical detection using either light scattering from a laser or otherfocused light source, impedance spectroscopy, fluorescence detection(assuming the cells were fluorescently tagged), or some form of imagingsoftware that was capable of counting cells based on size. This wouldfind utility in forensic applications defining when the requisite numberof sperm from the biological sample required for the analysis had beencollected in the collection reservoir.

In a further embodiment, the present invention is used to isolate spermfrom other cells or other biologically-derived molecular species viasome flow-driven separation process presents the possibility ofquantitating subpopulations of sperm from the sample. This couldfacilitate the evaluation of sperm that are dysfunctional with respectto fertilization ability, the separation of sperm subpopulations thatare functional relative to those that are dysfunctional due to exposureto toxicants (apoptotic) or cryostorage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the size difference between sperm and epithelial cells.

FIG. 2 outlines microchannel cell separation based on celldensity/adhesion differences.

FIG. 3 outlines microchannel cell separation in an electric field-drivensystem based upon density, proclivity for adhesion, and electrophoreticmobility. The sperm are swept with the flow to the cathodic reservoir(right).

FIG. 4 shows an alternate manifestation of the microchannel cellseparation in an electric field-driven system based upon density,proclivity for adhesion, and electrophoretic mobility. In thisthree-reservoir system, the cell mixture is deposited in the centralreservoir, and the epithelial cells and sperm cells are collected in theoutside reservoirs.

FIG. 5 shows the present invention being used as part of amulti-function (multiple ‘domain’) totally-integrated system.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention exploits physical and/or biological properties ofsperm and other biological materials, such as epithelial cells, toeffect a robust and reliable separation of the two cell types.Biological materials used herein includes, but is not limited to, othercells, such as epithelial cells, red blood cells, white blood cells,etc.; molecular species, such as nucleic acids (RNA and DNA), proteins,etc.; cell membranes; and organelles. Two separation approaches can beutilized to invoke separation of cells, with a main focus on theseparation of sperm from other cells for forensic analysis where boththe sperm and the other cells can be important in the forensic process.The first mode amenable to a microfabricated device is a separationdriven by an electric field—this inherently involves both anelectrophoretic component (mobility of cells based on size and theirsurface charge) and a flow component in the form of electroosmotic flow(EOF—the flow that results from the presence of ions in glass channel).The second type, one that does not invoke the use of electric fields butis based solely on flow, can be driven by a number means includinggravity-driven (siphoning), hydrostatic pressure (or vacuum)-drivenflow, or centrifugal driven flow.

I. Cell Separation Exploiting Electrokinetic Phenomena

In microchip electrophoresis, analytes are acted upon by two forces,intrinsic electrophoretic mobility (μ_(ep)), governed by thecharge-to-size ratio of the analyte, and EOF, generated by charge on themicrochannel surface. For cell separations, these forces can be employedtogether, or EOF can be reduced (or close to zero), with theelectrophoretic mobilities providing the main governing force for theseparation. Consequently, three scenarios emerge where separation isdriven by 1) electrokinetic phenomena specific to the cells themselves;2) a combination of electrokinetic phenomena specific to the cells andthe EOF; and 3) the low volume, plug-type flow resulting from EOF. Theseare addressed individually below.

A. Separation Based Solely on Cell Electrophoresis

The simplest scenario in microchips, one where the chip surface wastreated to negate the EOF, cell electrophoretic mobility becomes thedominant separation force. This has already been demonstrated in theliterature (Kricka et al., 1993) by the separation of live and deadsperm in an electric field, which is likely due to differences in thecell surface charge, however, the role of EOF in this separation cannotbe ruled out. For sperm and epithelial cells, the significant sizedifference (4-5 μm vs 50 μm) presents a scenario where there is likelyto be significant differences in charge-to-size ratio, and this may beexploited for the sake of separation (FIG. 1). In addition, the surfacecharge of the cells can be varied with pH, solution composition, andionic strength of the separation buffer. This allows altering thesurface charges in the electrophoretic-based separation scheme tooptimize the separation speed and efficiency.

An electrophoretically-driven system is attractive because, in additionto separation of the cells, there is a cellular concentrating effect.Therefore, the buffer volume used to desorb the biological material fromthe swab would have minimal impact on downstream sample preparation oranalytical processes where volume limitations may exist. In addition,any free DNA in the biological material is not captured in the spermfraction.

B. Separation Based on Cell Electrophoresis and EOF

In addition to exploiting cell electrophoretic mobility, a significantEOF provides a flow bulk component to the separation and, under theappropriate circumstances, can enhance the separation. Under conditionswith a reasonable EOF, the differential movement of sperm and epithelialcells exists under low electric field strengths (about 5-1000 V/cm,preferably about 25-300 V/cm, most preferably about 75-100 V/cm). Spermmigrate toward the cathode, while epithelial cells have an oppositemobility (to the anode). However, in the same way that the surfacecharge of the cells can be altered by the pH, solution composition, andionic strength of the separation buffer, so can the EOF. A high solutionionic strength reduces the charge on the microchannel surface (the zetapotential) and, hence, reduces the EOF. Reducing or even eliminating thecharge on the microchannel surface by covalent, dynamic or absorptivecoating can similarly reduce or minimize EOF. A similar effect can beachieved by reducing the solution pH, but this is less attractive withcells that will need to be maintained in the biological pH range.Consequently a number of approaches can be used to optimize the EOF thatallows for optimal separation of the analytes involved, in thisparticular case, different biological materials, specifically, sperm andepithelial cells.

C. Separation Based Solely on Electric Field-Driven Flow (EOF)

See EOF section below.

II. Flow-Based Separations

A critical aspect of this mechanism is the magnitude of the flow usedfor the separation. A flow that is low in magnitude (about 0.1-1000μL/hr, preferably about 0.3-10 μL/hr, and most preferably about 0.6μL/hr) and reproducibly-controlled flow is utilized for theseseparations and can be achieved with a number of approaches.

A. EOF

The low magnitude, plug-type flow associated with EOF (no turbulence) isideal for separating cells based on physical characteristics.Modification of the silica surface charge allows control of EOF andprovides a support for electrostatic interactions that can furtherincrease the cell separation efficiency. Under low electric fieldstrength (e.g., ˜33V per cm of microchannel), we have observed thedifferential movement of sperm and epithelial cells inphosphate-buffered saline (pH 7.4)—the sperm cells migrate to thecathode and epithelial cells migrated to the anode. Hence, placement ofa mixture of sperm and epithelial cells in a reservoir on a microdevice,and proper placement of electrodes results in the separation of spermcells from the mixture into another reservoir containing the cathode. Anapplied field is used to direct the sperm cells into the desiredreservoir on the microdevice (FIG. 3). The migration of epithelial cellsto the anode is due to their negative surface charge. Sperm cells alsohave an overall negative surface charge, but the sperm migrate towardthe cathode because the magnitude of electroosmotic flow is greater thanthe magnitude of the electrophoretic mobility of the sperm cells. In aseparation based upon EOF flow, we can also take advantage of the othermechanisms of separation described herein such as density differences,proclivity for adhesion to the microchannel surface as well as to othercells, as well as the electrophoretic mobility differences. In thismanner, the selectivity and efficiency of separation can be enhanced.

In an alternate embodiment of this concept, the mixture reservoir can beplaced between two reservoirs connected in a linear fashion by amicrochannel etched into the glass (FIG. 4). By placing electrodes inthese two outside reservoirs, the mixture in the center can be separatedand the two cell types and/or biological materials collected in theseparate outside reservoirs. It should be noted that, in eithermanifestation, the use of a separation using electrokinetic effects hasthe added benefit in that any DNA in the cell mixture from cells lysedprior to the separation is attracted to the anode and, thus, isseparated from the sperm cell fraction. This is particularly importantin forensic applications.

B. Gravity-Driven Flow

Gravity-driven flow (siphoning) can also provide a low magnitude flowthat can be controlled with some accuracy and, hence, could be employedto differentially move the cells in microchannels. Under theseconditions, the effect of gravity not only drives the flow of fluid fromone reservoir to the other, but density differences in the cells in amixture can be exploited, in which the epithelial cells settle morereadily than sperm cells. For example, in the case of sperm andepithelial cells, approximately 5 minutes is sufficient to allow theepithelial cells to ‘settle’ to the bottom of the reservoir/channelbefore flow is induced. Flow is then induced by mismatched liquidheights in connecting reservoirs. The data shows that the fluid flowrate remains constant at an acceptable magnitude for at least 10minutes, allowing adequate time for a cell separation where sperm wereobserved leaving the mixture reservoir at a rate of approximately 5sperm/sec.

C. Pressure (or Vacuum)-Driven Flow

More reproducible and controllable flow rates can be generated in apressure-driven system employing the appropriate volume syringes andpumps. This uses the same mechanism of separation as the gravity-drivenflow, but would provide greater opportunity for automation due to theexternal control of the flow rate. Clearly what was accomplished withgravity-driven flow could be achieved with this system but in a muchmore automatable manner.

III. Combined Separation

Techniques discussed above are can be used alone or in combination.Various combinations are appropriate for the present invention. Asuccessful separation typically utilizes both flow and electrokineticseparations. The following are non-limiting examples of combinedseparations that are appropriate for the present invention: 1)separation utilizing electrokinetic phenomena and pressure-driven flow;2) separation utilizing pressure-driven flow and EOF; and 3) separationutilizing electrokinetic phenomena, pressure-driven flow, and EOF.Further, gravity, vacuum-driven and centrifugally-driven flow can easilysubstitute for the pressure-driven flow discussed in the possiblecombined separation regimes.

IV. Other Considerations for Isolation of Sperm Cells from a BiologicalMixture

A. Surface Area-to-Volume Considerations

There are a number of channel design modifications that result in anincreased surface-to-volume ratio, which we believe will also increasethe separation efficiency.

These include placing microfabricated posts in the separation channel.In this way, the posts (separated by approximately 8 μm) act as aphysical filter allowing sperm cells to freely flow through thebarriers, while the epithelial cells are too large (Chen et al., 1998).They utilized filters of varying size (5-35 μm) to separate the cells(based solely upon cell size) prior to DNA extraction of each fraction.Wilding et al. (1998) used 7 μm-spaced barriers in microchannels toeffect a size-based separation of white and red blood cells. An s-curvechannel shape will create a similar increase in surface-to-volume ratiowithout the incorporation of posts. An alternate manifestation of thiscell separation invention involves the use of increasedsurface-to-volume ratios in conjunction with the electroosmotic,pressure-driven and gravity-flow in the microchannel to optimize theseparation efficiency resulting from various physical and/or biologicalcharacteristics of the cells such as proclivity for adhesion, size, anddensity.

B. Exploiting Differential Adhesion

An inherent biological characteristic of white blood cells (WBCs) istheir ability to adhere to surfaces in biological systems. Wilding etal. (1998) exploited this, trapping WBCs using a series of weir-typefilters, with efficient trapping relying on increasing thesurface-to-volume ratio and enhancing the opportunity for WBCs to bindto the channel surface. A similar phenomenon is exploited in the currentinvention where sperm and epithelial cell mixtures may be separated asthe epithelial cells adhere to each other and to glass microchannelsurfaces to a much greater extent than do sperm cells. This results fromthe larger surface/contact area of the typically flat epithelial cells.In addition to exploiting the high proclivity for adhesion of epithelialcells (to the glass surface and to other epithelial cells) in comparisonto sperm, the cell separation shown in FIG. 2 is also based upon theirsize and density. The sperm cells, smaller and less dense, are swept bythe fluid movement into the channel and to the outlet reservoir.

C. Capture of Free DNA and Other Non-sperm Components

The sperm separation method of the present invention may be optimized toeffectively remove other non-sperm components of the mixture that may beproblematic to the user. These components can include, but are notlimited to, DNA and other cells such as white blood cells, red bloodcells bacteria and yeast. DNA can be effectively prevented fromcontaminating the sperm cell fraction with the use of apositively-charged microchannel coating combined with the appropriatebuffer (possessing the appropriate ionic strength, pH, etc.), or withthe use of a buffer (possessing the appropriate ionic strength, pH,etc.) needed for use of a bare (untreated) microchannel wall. In asimilar manner, a positive, neutral, or negative microchannel coating(covalent or dynamic) may be needed in conjunction with the appropriatebuffer (ionic strength, pH, etc.) to optimize the separation of spermfrom other non-sperm components. In addition, the ionic strength, pH,concentration, and viscosity of the electrolyte solution may beoptimized by the addition of other modifiers (e.g., detergent) tooptimize the removal of unwanted cellular, protein, nucleic acid or lowmolecular weight components that may interfere with analysis.

V. Microfabricated Devices

Microfabricated or microfluidic devices are used to perform theseparation of the present invention. “Microfabricated” or“microfluidic,” as used herein, refers to a system or device havingfluidic conduits or microchannels that are generally fabricated at themicron to submicron scale, e.g., typically having at least onecross-sectional dimension in the range of from about 0.1 μm to about 500μm. The microfluidic system of the invention is fabricated frommaterials that are compatible with the conditions present in theparticular experiment of interest. Such conditions include, but are notlimited to, pH, temperature, ionic concentration, pressure, andapplication of electrical fields. The materials of the device are alsochosen for their inertness to components of the experiment to be carriedout in the device. Such materials include, but are not limited to,glass, quartz, silicon, and polymeric substrates, e.g., plastics,depending on the intended application.

The device generally comprises a solid substrate, typically on the orderof a few millimeters thick and approximately 0.2 to 12.0 centimeterssquare, microfabricated to define at least one inlet reservoir, at leastone outlet reservoir, and a microchannel flow system, preferably anetwork of flow channels, extending from the at least one inletreservoir to the at least one outlet reservoir. In the embodimentdepicted in FIGS. 2 and 3, a sperm containing biological sample isapplied to the inlet reservoir; and the sperm moves, under variousforce(s) discussed above, from the inlet reservoir through themicrochannel to the outlet reservoir.

In the embodiment depicted in FIG. 4, the device comprises at leastthree reservoirs and at least two channels. The inlet reservoir isconnected to a first outlet reservoir by a first channel, and isconnected to a second outlet reservoir by a second channel. A spermcontaining biological sample is applied to the inlet reservoir; and thesperm moves, by EOF and electrophoretic mobility, from the inletreservoir through the microchannel to the first outlet reservoir, whilethe other cells, preferably epithelial cells, moves from the inletreservoir to the second outlet reservoir.

Although the drawings show only one separation apparatus, multipleseparations may be accomplished on a single chip. These multiplexedseparations can be done in parallel or at different times, depending onthe load requirements of the user. Further, the main separation channelcan intersect and connect with other channels. This is important, forexample, for diluting the sample, adjusting the pH of the sample, addingreactants to the sample, coating the channel, etc. For the case ofadjusting the pH, the intersection can be used to inject acid and/orbase to the solution flowing in the main separation channel. In doingso, the pH of the solution flowing in the main separation channel can becontrolled and varied along the length of the channel.

Analytical devices having microfabricated flow systems can be designedand fabricated in large quantities from a solid substrate material. Theyare preferably easy to sterilize. Silica and silicon are the preferredsubstrate materials because of the well-developed technology permittingits precise and efficient fabrication, but other materials may be usedincluding cast or molded polymers including polytetrafluoroethylenes andpolydimethylsiloxane (PDMS). The sample inlet and other reservoirs, themicrofabricated flow system, including the flow channel(s) and otherfunctional elements, may be fabricated inexpensively in large quantitiesfrom a silicon substrate by any of a variety of micromachining methodsknown to those skilled in the art. The micromachining methods availableinclude film deposition processes such as spin coating and chemicalvapor deposition, laser fabrication or photolithographic techniques suchas UV or X-ray processes, or etching methods which may be performed byeither wet chemical processes or plasma processes.

Flow channels of varying widths, depths, and shape can be fabricatedwith microfluidic dimensions for use in sperm separation. The silicasubstrate containing a fabricated microchannel may be covered andsealed, e.g., thermally bonded, with a thin glass cover. Other clear oropaque cover materials may be used. Alternatively, two silica substratescan be sandwiched, or a silicon substrate can be sandwiched between twoglass covers. The use of a transparent cover results in a window whichfacilitates dynamic viewing of the channel contents, and allows opticalprobing of the micro-flow system either visually, by machine, and/or bylaser interrogation. Other fabrication approaches can also be used.

The capacity of the devices is very small and therefore the amount ofsample fluid required for an analysis is low. For example, in a 3 cm×3cm silicon substrate, having on its surface an array of 50 channelswhich are 120 μm wide×40 μm deep×2 cm (2×10⁴ μm) long, the volume ofeach groove is 0.096 μL and the total volume of the 50 grooves is 4.8μL. The low volume of the microfabricated flow systems allows assays tobe performed on very small amounts of a liquid sample (<5 μL). Thedevices may be microfabricated with microliter volumes, or alternativelynanoliter volumes or less, which advantageously limits the amount ofsample, buffer or other fluids required for an analysis. Thus, animportant consequence and advantage of employing flow channels havingmicroscale dimensions is that very small scale analyses can beperformed.

To provide appropriate electric fields, the system generally includes avoltage controller that is capable of applying selectable voltagelevels, sequentially or, more typically, simultaneously, to each of thereservoirs, including ground. Such a voltage controller is implementedusing multiple voltage dividers and multiple relays to obtain theselectable voltage levels. Alternatively, multiple independent voltagesources are used. The voltage controller is electrically connected toeach of the reservoirs via an electrode positioned or fabricated withineach of the plurality of reservoirs. In one embodiment, multipleelectrodes are positioned to provide for switching of the electric fielddirection in a microchannel, thereby causing the analytes to travel alonger distance than the physical length of the microchannel. Use ofelectrokinetic transport to control material movement in interconnectedchannel structures was described, e.g., in WO 96/04547 to Ramsey, whichis incorporated by reference.

Modulating voltages are concomitantly applied to the various reservoirsto affect a desired fluid flow characteristic, e.g., continuous ordiscontinuous (e.g., a regularly pulsed field causing the sample tooscillate direction of travel) flow of labeled components toward a wastereservoir. Particularly, modulation of the voltages applied at thevarious reservoirs can move and direct fluid flow through theinterconnected channel structure of the device.

Another way to control flow rates is through creation of a pressuredifferential. For example, in a simple passive aspect, a cell suspensionis deposited in a reservoir or well at one end of the channel, and atsufficient volume or depth, that the cell suspension creates ahydrostatic pressure differential along the length of the channel, e.g.,by virtue of its having greater depth than a well at an oppositeterminus of the channel. Typically, the reservoir volume is quite largein comparison to the volume or flow through rate of the channel, i.e., 1μL reservoirs or larger as compared to a 100 μm channel cross section.Another pressure based system is one that displaces fluid in themicrofluidic channel using, e.g., a probe, piston, pressure diaphragm,or any other source capable of generating a positive or negativepressure.

Alternatively, a pressure differential is applied across the length ofthe channel. For example, a pressure source is optionally applied to oneend of the channel, and the applied pressure forces the material throughthe channel. For example, pressure applied at the inlet reservoir wouldforce the cell mixture contained therein through the microchannel, andinto the outlet reservoir. The pressure is optionally pneumatic, e.g., apressurized gas or liquid, or alternatively a positive displacementmechanism, i.e., a plunger fitted into a material reservoir, for forcingthe material along through the channel. Pressure can, of course, also bedue to electrokinetic force, thermal expansion, or a variety of othermethods and devices.

Alternatively, a vacuum source (i.e., a negative pressure source) isapplied to a reservoir at the opposite end of the channel to draw thesuspension through the channel. A vacuum source can be placed in theoutlet reservoir to draw a cell suspension from the inlet reservoir.Pressure or vacuum sources are optionally supplied external to thedevice or system, e.g., external vacuum or pressure pumps sealablyfitted to the inlet or outlet of the channel, or they are internal tothe device, e.g., microfabricated pumps integrated into the device andoperably linked to the channel, such as those disclosed in WO 97/02357to Anderson et al., which is incorporated herein by reference.

Alternatively, flow in this system could be established by centrifugalforces generated by spinning microdevices around a central axis. Thechannels in the microdevices would be situated at least partly radiallyoutward from the central axis with the inlet reservoir closer to thecentral axis than the outlet reservoir. Spinning instrumentation (e.g.centrifuge) external to the microdevice would be used to generate therequired rotational motion. Flow rates through the microchannels wouldbe controlled by changing the speed of the rotation, the distance fromthe central axis, or both.

The microchip-based cell separator can be designed as a mono-taskingstand-alone unit that serves a single function—cell separation. Thiswould be consistent with the above discussion. With this system, cellsextracted or desorbed from the sampling instrument, such as cottonapplicator, would be added to the inlet reservoir in the appropriatevolume where application of the appropriate forces would used tofacilitate the cell separation. The separated material, sperm and othercells, would be removed from their respective reservoirs for subsequentanalysis.

The microchip-based cell separator can also be envisioned as part of amulti-function (multiple ‘domain’) totally-integrated system that cariesout numerous processes, either simultaneously or serially (FIG. 5). Thisinvolves the cell separator as only one of many domains in an integratedsystem that could provide ‘sample in/answer out’ capability. Thisarrangement has the cell separation domain receiving a cell mixture from‘upstream’ processing, via fluidic transfer, from a cell extraction(e.g., elution and/or desorption) domain where the cell mixture isobtained and removed from the original sampling instrument. Followingseparation of the sperms from other cells, the sperms and other cellsare transferred for downstream processing which involves fluidictransfer to one of two subsequent domains for processing. In oneembodiment, the sperms and/or others cells are transferred to a ‘DNAextraction’ domain and then to the ‘PCR’ domain for select target DNAamplification prior to STR typing. Alternatively, the sperms and/orother cells would be transferred directly to the PCR domain for selecttarget DNA amplification.

Such integrated system can be carried out with a ‘valveless’ microchipwhere control of fluidic movement is carried out with pumps orelectrokinetically. Alternatively, the use of a valved system can beinvoked. This integrated approach allows for insulation of each of thedomains more effectively and minimizes leakage or contamination ofreagents from one domain to another.

Although certain presently preferred embodiments of the invention havebeen specifically described herein, it will be apparent to those skilledin the art to which the invention pertains that variations andmodifications of the various embodiments shown and described herein maybe made without departing from the spirit and scope of the invention.Accordingly, it is intended that the invention be limited only to theextent required by the appended claims and the applicable rules of law.

1. A method for isolating sperm cells comprising the steps of a)providing a biological sample containing sperm cells; b) providing a atleast a first reservoir and a second reservoir, and a microchannelconnecting the first and second reservoirs; c) placing the sample intothe first reservoirs; d) applying a separation means between the firstand second reservoirs to separate the sperm cells from other biologicalmaterials; and e) collecting the sperm cells, substantially free of theother biological materials, in the second reservoir.
 2. The method ofclaim 1, wherein the separation means is an electric field.
 3. Themethod of claim 1, wherein a potential is placed between the first andsecond reservoirs.
 4. The method of claim 1, wherein the separationmeans is electroosmotic flow.
 5. The method of claim 1, wherein theseparation means is a pressure-induced flow.
 6. The method of claim 1,wherein the other biological materials are selected from the groupconsisting of epithelial cells, white blood cells, red blood cells,bateria, yeasts, proteins, RNAs, DNAs, and combinations thereof.
 7. Themethod of claim 1, wherein the separation means is electroosmotic flowand an electric field.
 8. The method of claim 7, wherein a potential isplaced between the first and second reservoirs.
 9. The method of claim1, wherein the separation means is electroosmotic flow andpressure-induced flow.
 10. The method of claim 9, wherein thepressure-induced flow is generated by gravity.
 11. The method of claim9, wherein the pressure-induced flow is generated by a pump.
 12. Themethod of claim 9, wherein the pressure-induced flow is generated by avacuum.
 13. The method of claim 9, wherein the pressure-induced flow isgenerated by rotational motion.
 14. The method of claim 1, furthercomprising a third reservoir connecting to the first reservoir via asecond microchannel.
 15. The method of claim 14, wherein a potential isapplied between the second and the third reservoir.
 16. The method ofclaim 1, wherein the separation means is electroosmotic flow, electricfield, and pressure-induced flow.
 17. The method of claim 16, whereinthe pressure-induced flow is generated by gravity.
 18. The method ofclaim 16, wherein the pressure-induced flow is generated by a pump. 19.The method of claim 16, wherein the pressure-induced flow is generatedby a vacuum.
 20. The method of claim 16, wherein the pressure-inducedflow is generated by rotational motion.
 21. The method of claim 1,further comprising a third reservoir connecting to the first reservoirvia a second microchannel.
 22. The method of claim 21, wherein apotential is applied between the second and the third reservoir.
 23. Themethod of claim 21, wherein the other biological materials migrate fromthe first reservoir to the third reservoir and the sperm cells migratefrom the first reservoir to the second reservoir.
 24. The method ofclaim 21, wherein the other biological materials are selected from thegroup consisting of epithelial cells, white blood cells, red bloodcells, bateria, yeasts, proteins, RNAs, DNAs, and combinations thereof.25. The method of claim 1, wherein the separation means ispressure-induced flow and an electric field.
 24. The method of claim 1,wherein the biological sample comes from a vaginal swab.
 25. The methodof claim 1, wherein the other biological materials are further analyzed.26. The method of claim 1, wherein the sperm cells are further analyzed.27. The method of claim 1, wherein at least one valve is present in themicrochannel for flow-control.
 28. The method of claim 1, wherein thesperm cells collected in the second reservoir is concentrated.